Volume3 no.1 January1976

Nucleic Acids Research

Specific spin-labeling of transfer ribonucleic acid molecules.

Marcel Caron and Hermann Dugas Department of Chemistry, University of Montreal, Montreal, Canada H3C 3V1

Received 8 September 1975 ABSTRACT The spin labels anhydride (ASL), bromoacetamide $BSL) and carbodiimide (CSLhwere used to label selectively tRNAGuu, tRNAfMet and tRNAke from E.coli. The preparation and characterization of the sites of la?beling of eight ne1 spin-labeled tRNAs are described. T e sites of labeling are: s U* using ASL, BSL and RL and tRNAG u; s4U using ASL and BSL on tRNAfMet and tRNA ; U-37 with CSL on tRNAfMet; U-33 with CSL on tRNAPhe The rare base X at position 47 of tRNAPhe has been acylated with a spin-labeled N-hydroxysuccinimide (HSL). The 3'end of unfractionated tRNA molecules has been chemically modified to a morpholino spin-labeled analogue (MSL). Their respective e.s.r. spectra are reported and discussed.

INTRODUCTION In the past five years, the spin-labeling technique has

been explored to study the conformational and fonctional properties of transfer RNA(tRNA) molecules. Rich and coworkers (1,2) have spin-labeled the a-amino group of Val-tRNAVal and PhetRNAPhe from E.coli and the sulphydryl group of Cys-tRNACYS (3) to study the temperature-induced conformational perturbations of the CCA end of these tRNA molecules. Latter, the group of Nishimura (4) succeeded in spin-labeling the s 4U residue located in the region between hU stem and CCA stem of various E.coli tRNAs. In a previous communication (5) we reported the selective labeling of the rare base s2U* at the wobble position of the anticodon of E.coli tRNAGlu using a spin label anhydride (ASL).

Abbreviations used: ASL, anhydride spin label; BSL, bromoacetamide spin label; CSL, carbodiimide spin label; HSL, N-hydroxysuccinimide spin label; MSL, morpholino spin label; s4U, 4-thiouridine; s2U*, 2-thio-5(N-methylaminomethyl) uridine; X, 3-(3-amino-3-carboxy-n-propyl) uridine. ¢ Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England

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Nucleic Acids Research These spin label experiments provided valuable informations about the motional freedom of the nitroxide moiety at or It would be extremely useful to prenear the site of labeling. pare other labeled-tRNA molecules with spin labels located at other strategiC positions of the molecule. It is exactly the goal that we had in mind when we started this project. The changes in spin mobility reflecting the behavior of a particular portion of the molecule of such tRNA derivatives would be of great value to monitor the structure-function relations in tRNA molecules. We present here the detailed preparation and characterization of eight new spin-labeled tRNAs using the four spin labels shown below: H 0

-

N

C\Y"NOC2H5

ASL OTs(

N=C =

o -N

N_CH2_JH1_N CH3

CSL

We have also labeled the 3'CCA end of unfractionated tRNA by carrying out the following transformations (6) of the terminal adenosine ribose ring:

o

tRNA--

Ad

1) NaIO4

tRNA__O

Ad

O

HO

OH

2) H2N

3)

NaBH4

N'N-°O

N

N

o

20

MSL

Nucleic Acids Research Some of the specific problems encountered in preparing such tRNA derivatives and the future potential usefulness of these molecules are discussed. EXPERIMENTAL Materials

Unfractionated tRNA from E.coli B was purchased from Plenum Scientific Research Inc. (Hackensack, N.J.). Purified tRNA from E.coli K-12 (tRNA fMet-lot.15-290, activity 100%; tRNAGlu-lot.15-291, activity 78%; tRNAPhe-lot.15-292, activity 69%) were kindly provided by Dr. A. Kelmers of the Oak Ridge National Laboratory. 3-(2-bromoacetamido)-2,2,5,5-tetramethyl pyrrolidine-loxyl (BSL) and 3-carboxy-2,2,5,5-tetramethyl pyrroline-l-oxyl anhydride with ethyl hydrogen carbonate (ASL) were obtained from Syva Corp. (Palo Alto, Calif.). 4-amino-2,2,6,6-tetramethyl piperidine-l-oxyl was prepared from 4-acetamido-2,2,6,6-tetramethyl piperidine-l-oxyl (Frinton Labs, S. Vineland, N.J.) according to Rosantzev (7). 1-(N-methyl-2-morpholinoethyl)-3-(2,2,6,6-tetramethyl piperidine-l-oxyl) carbodiimide p-toluenesulfonate (CSL) was synthesized following the procedure of Kumarev and Knorre (8). The N-hydroxysuccinimide ester of 3-carboxy-2,2,5,5 tetramethyl pyrrolidine-l-oxyl (HSL) was prepared according to the procedure of Anderson and F8lsch (9). DEAE-Sephadex A-25 was obtained from Pharmacia Chemicals (Montreal, Canada); Dowex 1-X2 (Cl ) resin was a product of BioRad Labs. (Richmond, Calif.); DEAE-cellulose was purchased from Schwarz-Mann (Orangeburg, N.Y.). RNase T1 was a product of Calbiochem. (San Diego, Calif.). RNase T2 and pancreatic RNase A ware obtained from Sigma (St-Louis, Miss.). Buffer solutions were made by dissolving the appropriate chemicals of the purest form available in doubly distilled deionized water.

Instrumentation The EPR spectra were recorded on a Bruker 414S spectrometer operating at 9.5 GHz and equipped with a rectangular cavity.

The microwave power was kept below 5 mW. The samples were taken into a small aqueous flat cell (Scanlon Co.). Absorbances were measured on a Perkin-Elmer Coleman 55 spectrometer. 21

Nucleic Acids Research Spin-labeling of tRNA a) using ASL and BSL The general procedure reported by Hara et al. (4) has been followed. A solution of 5.6 mg of spin label in 0.3 ml of methanol was added to a solution of 5.0 mg of tRNA in 5.0 ml of a buffer solution containing 5 mM Na2Co3 and 90 mM NaHCO3 (pH 8.9). The resulting mixture was stirred for 5 hrs at 250. The solution was then neutralized by addition of 1.0 M NaOAc (pH 5.0). The spin-labeled tRNA was recovered by precipitation from cold ethanol and finally taken up in 0.25 ml of a 20 mM TrisHC1 buffer (pH 7.5) containing 10 mM MgCl2. The tRNA solutions were stored frozen at -20° and attempts to separate the labeled from the unlabeled tRNA molecules have been so far unsuccessful. b) using CSL A solution of 5.0 mg of tRNA and 20 mg of the spin label in 1.0 ml of a 20 mM Tris-HCl buffer (pH 8.3) containing 10 mM MgCl2 was incubated at 370 for 17 hrs. (The reaction was also performed in 50 mM Tris-HCl (pH 8.3) without MgCl2 and gave the same result.) The resulting solution was then neutralized with 0.2 M NaOAc (pH 5.0) and the spin-labeled tRNA was recovered and stored as above. c) using MSL According to the procedure of I. Smith and B. Malchey (private communication), 100 mg of unfractionated E.coli tRNA was dissolved in 20 ml of a 1.0 M NaOAc buffer (pH 5.0) containThe solution was kept at 40 for 2 hrs. After ing 20 mM NaIO4. neutralization with 4.0 M NaOAc, the tRNA was precipitated from cold ethanol. The precipitate was then dissolved in 10 ml of a 0.2 M Na2Co3 buffer (pH 9.5) containing 10% DMSO and 160 mg of 4-amino-2,2,6,6-tetramethyl piperidine-l-oxyl was added. The resulting mixture was stirred at 00 for 90 min. Then 0.75 ml of a 0.6 M NaBH4 solution was added and the solution was stirred for 30 min., after which another 0.75 ml of the NaBH4 solution was added and the solution was stirred for an additional 30 min. The mixture was then exhaustively dialysed against doubly distilled deionized water and the spin-labeled tRNA was recovered and stored as above. d) using HSL 22

Nucleic Acids Research According to the procedure of Gillam et al (10) 20 mg of purified tRNAPhe (E. coli) was dissolved in 2.5 ml of a 50 mM NaOAc buffer (pH 4.5) containing 10 mM MgCl2. A solution of 60 mg of HSL in 0.5 ml of freshly distilled dioxane was added. The pH was then quickly adjusted to pH 8.0 with 1.0 N NaOH and the resulting solution was stirred at room temperature for 17 hrs. After neutralization with glacial acetic acid, the spin-labeled tRNAPhe was recovered and stored as above. Conditions for enzymatic digestions (11) All incubations were conducted at 370. For pancreatic RNase A digestions, 20 O.D. of tRNA and 70 pg of enzyme were dissolved in 0.5 ml of a 20 mM Tris-HCl buffer (pH 7.5) and were incubated for 16 hrs. For RNase T1 digestions, the mixture consisted of 20 O.D. of tRNA and 50 units of enzyme in 0.5 ml of a 50 mM Tris-HCl buffer (pH 7.5) and was incubated for 18 hrs. For RNase T2 digestions, 20 OoD. of tRNA were incubated for 6 hrs. with 2.5 units of enzyme in 0.1 ml of a 50 mM KH2PO4 buffer (pH 4.7).

Chromatography of tRNA digests RNase T1 and/or pancreatic RNase A digests of tRNA were chromatographied on DEAE-Sephadex A-25 in 7 M urea according to Rushizky et al. (12). In a typical experiment, the digest was applied by means of a peristaltic pump to a DEAE-Sephadex A-25 column (120 x 0.5 cm) preequilibrated with a 20 mM Tris-HCl buffer (pH 7.5) containing 7 M urea and 0.14 M NaCl. Elution was carried out with a linear gradient obtained with 500 ml each of 0.14 M NaCl and 0.70 M NaCl in the presence of 20 mM Tris-HCl (pH 7.5) and 7 M urea. The flow rate was approximatively 10 ml/hr and fractions of 5.2 ml were collected. RNase T2 digests of tRNA were chromatographied on Dowex 1-X2 (CHO2) according to Offengand et al. (13). In a typical procedure, the digest was applied by means of a peristaltic pump to a Dowex 1-X2 (CHO2) column (30 x 0.5cm) preequilibrated with a 10 mM NH4CHO2 buffer (pH 3.7). Blution of s U* was performed with a linear gradient established with 100 ml each of 10 mM and 40 mM NH4CHO2 (pH 3.7). The flow rate was maintained at 10 ml/hr and 3.5 ml fractions were collected.

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Nucleic Acids Research Desalting of chromatographic fractions Using the procedure reported recently (14), mono-,diand trinucleotides were desalted by means of Dowex 1-X2 (HCO3) resin and 2.0 M NH4HCO3. For higher oligonucleotides, DEAEcellulose was used instead of Dowex. Solutions containing salt free oligonucleotides were then concentrated in a rotary evaporator.

Thin layer chromatograpgy Analysis of oligonucleotides obtained from column chromatography were performed using,a two-dimensional thin layer chromatographic system (11). Oligonucleotide-s were'first hydrolysed by incubation at 370 for 6 hrs with 1.0 N NaOH (total volum 0.5ml). Commercial cellulose plates (Eastman #6065) were used. The plate was eluted in the first dimension with isobutyric acid-0.5 M NH4OH(5:3) and with isopropyl alcohol-conc. HCl-H20 (70:15:15) in the second. The U.V. absorbing spots were eluted from the plate with water. RESULTS

Using the procedures described in the.Experimental Section, E.coli tRNA fMet tRNAPhe and tRNA Glu were successfully spinlabeled.respectively with ASL, BSL and CSL. In addition, E.coli tRNAPhe was coupled to HSL and the 3' end terminal adenosine of unfractionated E.coli tRNA was transformed into MSL. All those. chemical modifications were conducted in conditions such.tha.t the conformational integrity of the different tRNA species is assumed to be preserved. ..The EPR spectra.obtained from these eleven spin-labeled tRNA molecules.are depicted.in Figure 1 and are typical of molecules carrying only one spin.label (1-5). In order to be Able to draw local conformational data from these spectra? it is obvious that the exact.location of the spin label on each tRNA molecule has to be known.. a) Characterization of ASL-, BSL- and CSL-tRNAfMet According to Hara et al. (4), BSL reacts specifically nucleotide s4U of E.coli tRNAs and the disappeathio the with 4 rance of the s U-characteristic absorbance at 330 nm is an indication of its modification. The,elution patterns obtained, from column chromatography of the RNase T1 digests of the three

spin-labeled tRNA Met 24

as well as that of the control native

Nucleic Acids Research CBL-S3JXA!'h

U-eN

Cslrti,NA'

38L-tRNAA

ABL-tMeA

COL-tRNA,'

3BL-ItRNA7

A*L-tRN,

t

Fig. 1

A5OL-t3NAF

"K-% tWA

I-

EPR spectra of spin-labeled tRNAs at -22 HC1 (pH 7.5) containing 10 mM MgCl2.

in 0.02 M Tris-

tRNA fMet are illustrated on Figure 2. As seep in the figure, both ASL- and BSL-tRNAfMet give the same elution profile in which there is no absorbance at 330 nm under peak 4, the relative intensity of which is strongly decreased as compared to same peak in the control pattern. On the other hand, a large increase in relative intensity is apparent in peak lb whose fractions, after desalting, yielded an EPR spectrum representative of a spin-labeled oligonucleotide (5). Analysis of this tRNA fragment by two-dimensional T.L.C. on a cellulose plate afforded the sequence GGGGs 4U in both cases. Consequently the residue s U-8 is the site of labeling in both ASL- and BSL-tRNAfMet. The CSL-tRNAfMet digest gives an elution profile (Fig. 2c) that is almost identical to that of the control except for the 25

Nucleic Acids Research

0

.10

.4

z

I .401 I

-0

.1a

.4

Ir

3

.901

I

0

.1IM

.4

Ir -J,

I I -C

FIRACTION NUMBBR

Fig. 2

Elution patterns of RNase T1 digest on DEAE-Sephadex A-25. A) native tRNAfMet; B) ASL-, BSL-tRNAfMet; C) CSLtRNAfMet. The arrow indicates the peak containing the spin-labeled material.

I

I

FRACTION

Fig. 3

26

NUM33R

Elution patterns of RNase T1 digest on DEAE-Sephadex A-25. Al native tRNAPhe; B) ASL-, BSL-tRNAPne. The arrow indicates the peak containing the spin-labeled material.

Nucleic Acids Research loss in relative intensity that is apparent in peak 11 and which is accompanied by an increase of about the same order of magnitude in relative intensity in peak 8. After desalting, the material obtained from peak 8 gave an EPR spectrum related to a spinlabeled oligonucleotide (5). Analysis of the material recovered from peak 11 of the control elution yielded the fragment Cm2UCAUAACCCG of the anticodon region of tRNAfMet (15). Such an analysis performed on the paramagnetic material obtained from peak 8 showed also the presence of that same anticodon fragment. Chang's results on the chemical modification of tRNAfMet (16) showed that there are two nucleotides in that macromolecule that are extensively reactive toward a carbodiimide reagent, namely U-37 in the anticodon and U-47 in the miniloop. Accordingly, we conclude from the above mentioned results that U-37 in the anti. No sign of labeling on codon is the location of CSL in tRNA U-47 has been found. b) Characterization of ASL-, BSL- and CSL-tRNAPhe The elution patterns afforded by chromatography of the RNase T1 1 digests of both ASL- and BSL-tRNAPhe along with that of ~~~Phe are shown in Figure 3. the control native tRNA The two derivatized tRNAPhe yield the same profile in which absorbance at 330 nm is absent as in the case of ASL- and BSLtRNAfMeto The argumentation used then still holds here and it is concluded that s 4U-8 is the site of labeling in both ASLP and BSL-tRNA Phe The case is however different with CSL-tRNA Figure . 4 shows the elution profile of a pancreatic RNase digest of the spin-labeled tRNAPhe together with that of the control native

tRNAPhe It is seen in the former that the almost complete disappearance of peak 6 coincides with an equivalent increase in relative intensity of peak 2. After desalting, the material recovered from peak 2 gave an EPR spectrum consistent with a spin-labeled oligonucleotide (5). Analysis of the paramagnetic fragment yielded Phe the sequence UGAAA*A* located in the anticodon region of tRNA (17). However, considering the normal mode of action of pancreatic RNase (18), this fragment should have been GAAA*A*. The presence of an uridine (U-33) at the 5' end of this fragment can only

27

Nucleic Acids Research

0.2-

-0

1

0.2

0.2-

= k _

Specific spin-labeling of transfer ribonucleic acid molecules.

Volume3 no.1 January1976 Nucleic Acids Research Specific spin-labeling of transfer ribonucleic acid molecules. Marcel Caron and Hermann Dugas Depar...
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